Biology subjects

King, J. J.

Publications and source records attributed to King, J. J..

4 recordsLinked to original sources

bioRxiv2025

AID/APOBEC3 Dynamic Catalytic Pockets and AID-α7 Regulation Mechanisms

AID/APOBEC3s mutate dC to dU in ssDNA in the restriction of viruses, creation of anti-bodies, and when off-target affecting cancers. Little was known of the catalytic pocket dynamics of these enzymes in real time. As such, we utilised molecular dynamics analyses to determine catalytic pocket volumes and states. We found that most AID/APO-BECs remain predominantly in either a closed or indeterminate state to varying degrees while A3G-CD2 is predominantly open. This occurs chiefly from the catalytic residues themselves and the loop 1 serine/threonine in the "floor" of the catalytic pocket while other residues of the secondary catalytic loops 1, 3, 5, and 7 aid in these states. Surprisingly, we found that the presupposed stable position of AIDs unique 7 ({beta}-state) in contact with the {beta}-sheet and 6 was unstable while the -state in contact with 6 and loop7 was stable and caused pocket closure. Mutations could cause the destabilisation of the -state (R171Y and R178D) or stabilisation of the {beta}-state (R190D and R194A). Thus, pocket states are relatively similar in overall cause; however, multiple residues from secondary catalytic loops as well as AID-7 determine the rates at which these states occur, potentially affecting the chances any AID/APOBEC is able to have catalysis.

biochemistry↗

bioRxiv2025

Affinity-selected peptide ligands specifically bind i-motif DNA and modulate c-Myc gene expression

c-Myc is an oncogene that is dysregulated in ~70% of cancers. Its multifaceted function complicates effective drug targeting of the protein. i-Motif DNA structures in gene promotor regions have gained attention for their potential role in modulation of gene expression. These include the i-motif formed by the cytosine-rich sequence that lies upstream of the key P1 promotor of the c-Myc gene. Currently, selective ligands interacting with i-motif structures are limited. Here, peptide ligands for the i-motif from the promoter of c-Myc were identified via phage display. Hit peptides were filtered for selective binding to i-motif structures over other DNA structures using displacement assays and DNA melting experiments. Two lead peptides were found to produce dose-dependent changes on c-Myc gene expression after delivery into HEK293 cells expressing a c-Myc luciferase reporter construct. These leads may be used as chemical tools for the manipulation of c-Myc i-motif in vitro and have potential to be developed into cell-permeable peptidomimetics for delivery in vivo. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/656635v2_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@18a4e9aorg.highwire.dtl.DTLVardef@12e72b0org.highwire.dtl.DTLVardef@6ba866org.highwire.dtl.DTLVardef@1fcd338_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2023

Polymer-mediated oligonucleotide delivery enables construction of barcoded 3D cultures for spatial single-cell analysis

Spatial transcriptomics has been widely used to capture gene expression profiles, realised as a two-dimensional (2D) projection of RNA captured from tissue sections. Wree-dimensional (3D) cultures such as spheroids and organoids are highly promising alternatives to oversimplified and homogeneous 2D cell culture models, but existing spatial transcriptomic platforms do not currently have sufficient resolution for robust analysis of 3D cultures. We present a transfection-based method for fluorescent DNA barcoding of cell populations, and the subsequent construction of spheroidal cellular architectures using barcoded cells in a layer-by-layer approach. For the first time, changes in gene expression throughout this 3D culture architecture are interrogated using multiplex single-cell RNA sequencing in which DNA barcodes are used to encode the spatial positioning of cells. We show that transfection with fluorophore-conjugated barcode oligonucleotides enables both imaging and sequencing at single-cell resolution, providing spatial maps of gene expression and drug response. Additionally, we show that fluorophore-conjugated DNA barcodes support correlative imaging studies such as mechano-microscopy to capture information about spatially-varying mechanical heterogeneity in 3D cultures. We ability to create customised, encoded cellular assemblies is a general approach that can resolve spatial differences in gene expression in 3D cell culture models.

bioengineering↗

bioRxiv2023

Metastable Intermediates Identified in Epithelial to Mesenchymal Transition are Regulated by G-Quadruplex DNA Structures

Cancer is a heterogenous disease, with multiple cellular subpopulations present within a single tumour mass that differ genetically and morphologically, and thus respond differently to chemotherapeutics. Epithelial-to-Mesenchymal transition (EMT) has been shown to play a role in tumour heterogeneity. Single-cell sequencing is critical to identify cell-type-specific transcriptomic differences with multiplexing methods increasing experimental scope with reduced cost. Cell hashing with barcoded antibodies is commonly used to multiplex samples but is limited to samples expressing target antigens. Antigen-independent methods of barcoding cells, such as barcoded lipid-anchors, have gained traction but present substantial populations that cannot be unambiguously demultiplexed. Herein we report a multiplexed single-cell transfection-enabled cell hashing sequencing (scTECH-seq) platform, which uses antigen-independent endocytic uptake to barcode cells, resulting in efficient, uniform barcoding with high cell recovery. We apply this methodology to identify distinct metastable cell states in human mammary cells undergoing EMT and show that stabilisation of G-quadruplex DNA has the potential to inhibit EMT.

genomics↗